Project Summary Cancer progression is partly regulated by growth factors and their intracellular signaling networks. Healthy cells control growth factor signaling by internalization and subsequent recycling or destruction of the growth factor receptor in a ligand-dependent fashion. Recently, we found that the small molecule lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) presents a sufficient signal to internalize growth factor receptors in the absence of a ligand. PIP3 is one of the very first small molecules known to induce growth receptor internalization specifically which is of interest for treating cancer. Here, we will elucidate the mechanism of how PIP3 causes the internalization of epidermal growth factor receptor (EGFR). We will use a variety of unique chemical biology tools to acquire mechanistic answers to a number of hypotheses. The first hypothesis is that PIP3 binds directly to EGFR and induces endocytosis. In Aim 1, we will therefore synthesize a membrane-permeant, caged, photo- crosslinkable and clickable derivative of PIP3. Our lab has already prepared similar PIP3 derivatives in the past and has synthesized several of the key building blocks. The PIP3 derivative will be delivered to cells via bioactivatable groups, uncaged by light to induce binding and then photo-crosslinked to any binding protein. Mass spectrometry will demonstrate the intracellular generation of biologically active lipid species. Via click chemistry to affinity probes, we will extract the lipid-protein conjugates and perform proteomic analysis with a focus on known growth factor receptors. The second hypothesis is that the receptor is binding to an effector protein that primes it as cargo for endocytosis via clathrin-coated pits. We will prepare a number of truncated fluorescently labeled mutants to identify the minimal intracellular epitope of the receptor required for endocytosis (Aim 2). In the absence of a ligand and tyrosine phosphorylation, we will focus on Ser and Thr residues that might serve as anchoring points for protein binding. In order to avoid interference of the fluorescent label with the endocytic machinery, we will use genetic code expansion to introduce fast reacting amino acids for minimally invasive labeling. Preliminary data demonstrated the feasibility of this technique to follow receptor internalization by confocal microscopy. Therefore, the third hypothesis is that specific Ser and Thr residues are phosphorylated by the MAP kinase p38. We will demonstrate EGFR phosphorylation and its inhibition in vitro and in cells. To demonstrate functional relevance, we will prepare a p38 construct that can be switched on by adding a small molecule (a chemical dimerizer) that translocates the enzyme to the plasma membrane (Aim 3). Once successful, we will use similar constructs to translocate proteins that we already identified by an RNAi screen as essential for PIP3-induced endocytosis, e.g. PAR3 & PAR6. As a readout, we will use the fluorescently labeled receptor variants from Aim 2 in live cells. The combined results will help to better understand receptor internalization mechanisms in the absence of a ligand. Any protein essential for this process must be considered a prime target for novel therapeutics to reduce cell surface growth factor receptor levels and cancer progression.